Bacillus methanolicus pyruvate carboxylase and homoserine dehydrogenase I and II and their roles for l-lysine production from methanol at 50°C

We here present the pyc gene encoding pyruvate carboxylase (PC), and the hom-1 and hom-2 genes encoding two active homoserine dehydrogenase (HD) proteins, in methylotrophic Bacillus methanolicus MGA3. In general, both PC and HD are regarded as key targets for improving bacterial l-lysine production; PC plays a role in precursor oxaloacetate (OAA) supply while HD controls an important branch point in the l-lysine biosynthetic pathway. The hom-1 and hom-2 genes were strongly repressed by l-threonine and l-methionine, respectively. Wild-type MGA3 cells secreted 0.4 g/l l-lysine and 59 g/l l-glutamate under optimised fed batch methanol fermentation. The hom-1 mutant M168-20 constructed herein secreted 11 g/l l-lysine and 69 g/l of l-glutamate, while a sixfold higher l-lysine overproduction (65 g/l) of the previously constructed classical B. methanolicus mutant NOA2#13A52-8A66 was accompanied with reduced l-glutamate production (28 g/l) and threefold elevated pyc transcription level. Overproduction of PC and its mutant enzyme P455S in M168-20 had no positive effect on the volumetric l-lysine yield and the l-lysine yield on methanol, and caused significantly reduced volumetric l-glutamate yield and l-glutamate yield on methanol. Our results demonstrated that hom-1 represents one key target for achieving l-lysine overproduction, PC activity plays an important role in controlling l-glutamate production from methanol, and that OAA precursor supply is not a major bottleneck for l-lysine overproduction by B. methanolicus.